MiR-9-5p and miR-106a-5p dysregulated in CD4+ T-cells of multiple sclerosis patients and targeted essential factors of T helper17/regulatory T-cells differentiation

Document Type: Original Article

Authors

1 Molecular Genetics Department, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

2 Division of Cellular and Molecular Biology, Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran

3 Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran

4 Institute for Nanoscience and Nanotechnology, Sharif University of Technology, Tehran, Iran

Abstract

Objective(s): Multiple sclerosis (MS) is considered as a chronic type of an inflammatory disease characterized by loss of myelin of CNS.Recent evidence indicates that Interleukin 17 (IL-17)-producing T helper cells (Th17 cells) population are increased and regulatory T cells (Treg cells) are decreased in MS. Despite extensive research in understanding the mechanism of Th17 and Treg differentiation, the role of microRNAs in MS is not completely understood. Thereby, as a step closer, we analyzed the expression profile of miR-9-5p and miR-106a-5p, and protein level of retinoic acid receptor (RAR)-related orphan receptor C (RORC; Th17 master transcription factor) as direct target of miR-106a-5p and forkhead box P3 (FOXP3; Treg master transcription factor) as indirect target of miR-9-5p in CD4+ T cells in two groups of relapsing and remitting in our relapsing-remitting MS (RR-MS) patients.
Materials and Methods:Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was utilized to assess the expression of miRNAs and mRNAs, in 40 RR-MS patients and 11 healthy individuals. Thus, FOXP3 and RAR-related orphan receptor γt (RORγt) was assessed in CD4+T-cells by flow cytometry. We also investigated the role of these miRNAs in Th17/Treg differentiation pathway through bioinformatics tools.
Results: An up-regulation of miR-9-5p and down-regulation of miR-106a-5p in relapsing phase of MS patients were observed compared to healthy controls. RORC and FOXP3 wereup-regulated in relapsing and remitting phases of MS, respectively.
Conclusion: Expression pattern of miR-9-5p and miR-106a-5p and their targets suggest a possible inducing role of miR-9-5p and suppressing role of miR-106a-5p in differentiation pathway of Th17 cells during MS pathogenesis.

Keywords

Main Subjects


1. Du C, Liu C, Kang J, Zhao G, Ye Z, Huang S, et al. MicroRNA miR-326 regulates TH-17 differentiation and is associated with the pathogenesis of multiple sclerosis. Nat Immunol 2009; 10:1252-1259.

2. McFarland HF, Martin R. Multiple sclerosis: a complicated picture of autoimmunity. Nat Immunol 2007; 8:913-919.

3. Lassmann H, Brück W, Lucchinetti CF. The immunopathology of multiple sclerosis: an overview. Brain Pathol 2007; 17:210-218.

4. Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 Cells. Annu Rev Immunol 2009; 27:485-517.

5. Dong C. TH17 cells in development: an updated view of their molecular identity and genetic programming. Nat Rev Immunol 2008; 8:337-348.

6. Harrington LE, Hatton RD, Mangan PR, Turner H, Murphy TL, Murphy KM, et al. Interleukin 17–producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol 2005; 6:1123-1132.

7. Lassmann H, Brück W, Lucchinetti C. Heterogeneity of multiple sclerosis pathogenesis: implications for diagnosis and therapy. Trends Mol Med 2001; 7:115-121.

8. Park H, Li Z, Yang XO, Chang SH, Nurieva R, Wang Y-H, et al. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol 2005; 6:1133-1141.

9. Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B. TGFβ in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity 2006; 24:179-189.

10. Brucklacher-Waldert V, Stuerner K, Kolster M, Wolthausen J, Tolosa E. Phenotypical and functional characterization of T helper 17 cells in multiple sclerosis. Brain 2009; 132:3329-3341.

11. Ivanov II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A, Lafaille JJ, et al. The orphan nuclear receptor RORγt directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 2006; 126:1121-1133.

12. Yang XO, Pappu BP, Nurieva R, Akimzhanov A, Kang HS, Chung Y, et al. T helper 17 lineage differentiation is programmed by orphan nuclear receptors RORα and RORγ. Immunity 2008; 28:29-39.

13. Yang XO, Panopoulos AD, Nurieva R, Chang SH, Wang D, Watowich SS, et al. STAT3 regulates cytokine-mediated generation of inflammatory helper T cells. J BIOL CHEM 2007; 282:9358-9363.

14. Baecher-Allan C, Viglietta V, Hafler DA, editors. Human CD4+ CD25+ regulatory T cells. Semin Immunol; 2004; 16:89-98.

15. Zheng Y, Josefowicz SZ, Kas A, Chu T-T, Gavin MA, Rudensky AY. Genome-wide analysis of Foxp3 target genes in developing and mature regulatory T cells. Nature 2007; 445:936-940.

16. Fantini MC, Becker C, Monteleone G, Pallone F, Galle PR, Neurath MF. Cutting edge: TGF-β induces a regulatory phenotype in CD4+ CD25− T cells through Foxp3 induction and down-regulation of Smad7. J Immunol 2004; 172:5149-5153.

17. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. cell 2005; 120:15-20.

18. Boehm M, Slack FJ. MicroRNA control of lifespan and metabolism. Cell Cycle 2006; 5:837-840.

19. Hatfield S, Ruohola-Baker H. microRNA and stem cell function. Cell Tissue Res 2008; 331:57-66.

20. Negrini M, Ferracin M, Sabbioni S, Croce CM. MicroRNAs in human cancer: from research to therapy. J Cell Sci 2007; 120:1833-1840.

21. Pauley KM, Cha S, Chan EK. MicroRNA in autoimmunity and autoimmune diseases. J Autoimmun 2009; 32:189-194.

22. Organization WH. Atlas. Multiple sclerosis resources in the world, 2008: WHO; 2008.

23. Fenoglio C, Cantoni C, De Riz M, Ridolfi E, Cortini F, Serpente M, et al. Expression and genetic analysis of miRNAs involved in CD4+ cell activation in patients with multiple sclerosis. Neurosci Lett 2011; 504:9-12.

24. Dweep H, Sticht C, Pandey P, Gretz N. miRWalk–database: prediction of possible miRNA binding sites by “walking” the genes of three genomes. J Biomed Inform 2011; 44:839-847.

25. Hsu S-D, Lin F-M, Wu W-Y, Liang C, Huang W-C, Chan W-L, et al. miRTarBase: a database curates experimentally validated microRNA–target interactions. Nucleic Acids Res 2010;39:163-169.

26. Tesmer LA, Lundy SK, Sarkar S, Fox DA. Th17 cells in human disease. Immunol Rev 2008; 223:87-113.

27. Junker A, Krumbholz M, Eisele S, Mohan H, Augstein F, Bittner R, et al. MicroRNA profiling of multiple sclerosis lesions identifies modulators of the regulatory protein CD47. Brain 2009; 132:3342-3352.

28. Gandhi R, Healy B, Gholipour T, Egorova S, Musallam A, Hussain MS, et al. Circulating microRNAs as biomarkers for disease staging in multiple sclerosis. Ann Neurol 2013; 73:729-740.

29. Cox MB, Cairns MJ, Gandhi KS, Carroll AP, Moscovis S, Stewart GJ, et al. MicroRNAs miR-17 and miR-20a inhibit T cell activation genes and are under-expressed in MS whole blood. Plos One 2010; 5:e12132.

30. Paraskevi A, Theodoropoulos G, Papaconstantinou I, Mantzaris G, Nikiteas N, Gazouli M. Circulating MicroRNA in inflammatory bowel disease. J Crohns Colitis 2012; 6:900-904.

31. Fasseu M, Tréton X, Guichard C, Pedruzzi E, Cazals-Hatem D, Richard C, et al. Identification of restricted subsets of mature microRNA abnormally expressed in inactive colonic mucosa of patients with inflammatory bowel disease. Plos One 2010; 5:e13160.

32. Sonkoly E, Wei T, Janson PC, Sääf A, Lundeberg L, Tengvall-Linder M, et al. MicroRNAs: novel regulators involved in the pathogenesis of psoriasis? Plos One 2007; 2:e610.

33. Ouyang W, Beckett O, Ma Q, Paik J-h, DePinho RA, Li MO. Foxo proteins cooperatively control the differentiation of Foxp3+ regulatory T cells. Nat Immunol 2010; 11:618-627.

34. Chung CD, Liao J, Liu B, Rao X, Jay P, Berta P, et al. Specific inhibition of Stat3 signal transduction by PIAS3. Science 1997; 278:1803-1805.

35. Yagil Z, Nechushtan H, Kay G, Yang CM, Kemeny DM, Razin E. The enigma of the role of protein inhibitor of activated STAT3 (PIAS3) in the immune response. Trends Immunol 2010; 31:199-204.

36. Jetten AM. Retinoid-related orphan receptors (RORs): critical roles in development, immunity, circadian rhythm, and cellular metabolism. Nucl Recept Signal 2009; 7: e003.

37. Hu H, Djuretic I, Sundrud MS, Rao A. Transcriptional partners in regulatory T cells: Foxp3, Runx and NFAT. Trends Immunol 2007; 28:329-332.

38. Christopher AF, Kaur RP, Kaur G, Kaur A, Gupta V, Bansal P. MicroRNA therapeutics: Discovering novel targets and developing specific therapy. Perspect Clin Res 2016; 7: 68-74.